Despite identification of more than twenty ALS-associated genes (at least half of which also cause the second most frequent dementia, frontotemporal dementia or FTD), the disease mechanism(s) are unknown. Exploiting transgenic and gene targeted mice we have constructed, as well as cell culture models, which we have established with prior support from this grant application, we now propose a multi PI effort comprised of three interrelated directions, each designed to uncover disease mechanism(s) underlying inherited and sporadic ALS/FTD. First, in light of emerging genetic evidence for incomplete disease penetrance from single mutations in C9orf72 and TDP-43 and identification of two or more ALS/FTD-linked gene mutations in multiple sporadic ALS or FTD patients, we will test the hypothesis of oligogenic mutant gene synergy as a cause/contributor of apparently sporadic ALS or FTD through use of multiple mouse lines each of which develops only partial ALS- or FTD-like disease. Similarly, recognizing that heterozygous missense or truncation mutants of Tank-binding kinase 1 (TBK1) have been identified as causative of ALS/FTD, we will use our newly generated mouse models to determine how TBK1 contributes to motor neuron health and the consequences of reduction of its activity, including determination of whether reduction in TBK1 synergizes with other ALS-linked mutations to exacerbate motor neuron or cognitive disease in existing mouse lines that develop only partial ALS- or FTD-like disease from TDP-43 or C9orf72 transgenes alone. Finally, the discoveries of 1) liquid-liquid de-mixing as a mechanism for intracellular compartmentalization and 2) that such repetitive phase separation can nucleate protein misfolding are seminal findings of extraordinary biological interest and ones that have profound implications for neurodegenerative diseases. Recognizing this, we will exploit three cell culture models that we have established in which de-mixing can be induced to identify determinants, including the role of TBK1, of intracellular TDP-43 liquid-liquid de-mixing and aggregation, and to determine if there is cell-to-cell transmission of aggregated TDP-43 and if so initiate tests of the mechanism(s) of intercellular transmission.